Thyrotropin (TSH) pituitary glycoprotein hormone, plays a critical role in thyroid hormone production and thus in normal metabolism. TSH consists of an alpha subunit common to all glycoprotein hormones, and the unique TSHbeta subunit which is under more stringent physiological regulation. We have previously demonstrated that the hypothalamic peptide thyrotropin- releasing hormone (TRH) stimulates TSHbeta gene transcription in rat pituitary cell cultures, and portions of the 5'-flanking region of the gene confer TRH-stimulated responses to reporter genes in transient expression assays. Our first specific aim is to identify the specific TSHbeta gene elements which confer TRH responsiveness to luciferase constructs in GH3 and cultured pituitary cells. Bal 31 digestions, linker-scanner and point mutations of the gene will be performed, and the exact positioning of relevant DNA elements identified. Transient expression experiments will determine if TRH-sensitive gene regions colocalize with calcium and kinase C responses, and the effect of calcium on basal and TRH-stimulated TSHbeta gene transcription in normal pituitary cells will be measured. The second specific aim is to identify and isolate pituitary DNA-binding proteins which confer TRH stimulation to the TSHbeta gene. Binding of nuclear proteins to the TSHbeta gene will be assessed by DNAse I or Exonuclease III footprinting studies. Specific binding between defined gene elements and transcription factors will be measured by gel mobility shift assays and Southwestern blot analysis. Affinities of DNA-protein interactions will be calculated by Scatchard analysis and by competition and equilibrium binding studies. Because a transcription factor termed Pit-1/GHF-1 is thought to play a role in TRH stimulation of prolactin, DNA binding and transfection experiments with Pit-1 expression vector and the TSHbeta gene will be performed. These studies will determine if Pit-1 binds to the TSHbeta gene and assesses its potential role in TRH stimulation of TSHbeta. Transcription factors involved in the TRH response will be cloned from expression vector libraries by screening for DNA-binding proteins with specific TSHbeta gene enhancer oligonucleotides. The potential biological role of these proteins will be evaluated by cotransfection experiments using cloned factors and TRH receptor cDNA. Alternatively, protein factors can be isolated by chromatography on DNA enhancer oligonucleotide affinity columns. The combination of these approaches will directly define the DNA and proteins necessary to implement the TRH transcriptional response, and will provide important insight into the mechanism by which membrane-acting hormones exert effects at the gene level.